248
COUNSELI,, KLIMSTRA, AND COLTON
[CONTRIBUTION FROM
THE
VOL.
DIVISIONOF CHEMICAL RESEARCH, G.
I).
27
SEARLE & Co.]
Anabolic Agents. Derivatives of 5a-Androst-1-ene R. E. COUNSELL, P. D. KLIMSTRA,
AND
F. B. COLTON
Received July 86, 1961 Various derivatives of 5a-androst-1-ene have been prepared in the hope of obtaining compounds with high anabolic and minimal androgenic activity. The initial high order of biological activity in this series prompted the synthesis of other modifications. The preparation of 2a-methyl and 6&methyl-5a-androst-l-ene derivatives as well as a new method for the synthesis of the intermediate 2a-methyl-3-ketosteroids is described. Anabolic arid androgenic activities of a number of these compounds are reported.
As early as 1935 Kochakian and Murlin' demonstrated that testosterone had a marked affect on protein biosynthesis. The androgenicity of testosterone, however, has limited its clinical use for protein anabolic effects. As a conscquence, there has been a considerable effort to synthesize potent anabolic agents possessing little or no virilizing activity. * In 1940 Butenandt and Dannenberg3 reported that the A1 isomer of testosterone (IIa) was much less androgenic than testosterone itself. No information, however, has appeared pertaining to the myotrophic activity of this compound. Since this compound possessed all of the structural features generally associated with high anabolic activity, a program was initiated to prepare various substitu ted 5 a-androst-1-enes. m i r e 17p-hydroxy-,5a-androst-l-en-3-0ne (IIa) could be prepared readily by bromination of 5aandrostan-17p-ol-3-one and subsequent dehydrobromination, preparation of the 17a-alkyl homologs necessitated bromination under buffered conditions in order to keep the acid labile tertiary hydroxyl group intact. Bromination in an acetic acid-sodium acetate buffer or dimethylformamide was found to be suitable for this purpose. Moreover, the synthesis of the 3-keto-A'steroids was further complicated by the fact that dehydrohalogenation of the intermediate 2-bromocompounds gave some A4 isomer as a contaminant. This was not unexpected since Djerassi and Schold have shown that dehydrohalogenation of 2abromo-5a-androstan-17P-ol-3-one hexahydrobenzoate with collidiiie gave 42% and 18% of the A1 and A 4 isomers, respectively. We have also found that dchydrobromination of pure 2a-bromo-17a-methyl5a-androstan-17fi-ol-3-one with lithium chloride and (1) C. D. Kochakian and J. R. Murlin, J . Nutrition, 10, 437 (1935). (2) Cf. L. F. Fieser and M. Fieser, Steroids, Reinhold, New York, 19.59,p. 592. (3) A. Butenandt and H. Dannenberg, Bel.., 7 3 , 206 ( I 940). ( 4 ) The 1957 IUPAC rules on steroid nomenclature a8 set forth in J . .4m. Chem. Soc., 82, 5577 (1960), have been
followed.
L. ( 5 ) C . Djerami and C. R. Scholz, J . Am. Chem. Soc.: 69,
2404 (1947).
a. R1 = R2 = H b. R' = CH,, R2 = H C. R' = C~HI;. R2 = H d. R' = H, Ii'l = CH, e. R' = R2 = CHa f. R' = CzHs, R2 = CH, g. R1 = CHs, R2 = Br le
Ib
OR' I
C H3
A
c. ti. e. f. g.
R' R' R1 R' R'
= = = =
=
lithium carbonate in dimethylformamide gives both isomers. Thin layer chromatography of the crude dehydrobrominated product demonstrated the presence of approximately 5-10y0 of methyl testosterone.e Although the 3-keto- Al-steroids can be purified by chromatography, a more expedient method was sought. A modification of the recently reported' use of sodium metabisulfite for purification of isomeric corticoid derivatives was found to be particularly suitable and also obviated initial purification of the 2-bromo compound. In this process, advantage was taken of the differences in the rate of reaction of the various products with bisulfite ion. Brief treatment (two to three minutes) of the crude dehydrohalogenated mixture with excess bisulfite in aqueous methanol a t room ~
~~
(6) We are indebted to Dr. E. G. Daskalakis and Mrs. J. Beilstein of our chromatography section for this data. (7) A. G. Long and L. J. Wyman, U. S. Patent 2,908,695 (1959).
JANUARY
1962
DERIVATIVES OF
temperature furnished the water soluble adduct of any unchanged saturated 3-ketosteroid. The less reactive unsaturated derivatives were removed by extraction and the residue obtained after removal of the solvent was retreated with bisulfite for one and one-half hours at the reflux temperature. This afforded a water-soluble adduct which upon alkaline decomposition gave pure 3-keto-A' material. Chromatography of the water insoluble residues showed that 2-bromo-3-keto-A', 3-ketoand 3-keto-A4-androstene derivatives were resistant to bisulfite-adduct formation under these conditions. When it was found that the 5a-androst-1-ene derivatives exhibited high anabolic activity, our study was expanded to include modifications of these molecules in the hope of potentiating this effect. The C-3 carbonyl group was reduced to the 3/3-01 with either lithium aluminum hydride or lithium tri-t-butoxyaluminohydride. The stereospecific course of hydride reduction of 3-keto-A'steroids to the 3p-alcohols is well established.8 The corresponding acetates were also prepared for biological evaluation. Since methyl substituents a t C-g9 and C-6lo in the androstane series have been reported to increase the anabolic-androgenic ratio, these modifications were made in the 5a-androst-l-ene series. The 2methyl analogs" were prepared from the corresponding 2a-methyl-5a-androstane derivatives. Ringold and coworkers9 have reported recently on the preharation of these intermediates by two general methods. One method involved condensation of the saturated 3-ketosteroid with excess ethyl oxalate and methylation of the resulting 2-ethoxgoxalate followed by reversal of the oxalate condensation with alkoxide. The other method, which they found to be more convenient, consisted of catalytic hydrogenation of the 2-hydroxymethylene derivatives. We have found that these compounds can also be prepared by hydrogenolysis of the 2a-dimethylaminomethyl derivative readily obtained via the Mannich reaction on the 3-ketosteroid progenitor. For example, hydrogenation of 2a-dimethylaminomethyl- l7a-methyl- 5a - androstan-17p-ol-3-one (IV) in isopropyl alcohol a t 130' under pressure over palladium-on-barium sulfate afforded after chromatography 71.5% yield of 2a,l7a-dimethyl-5a-androstan-17fl-ol-3-one (Ie), The 6p-methyl analogs were prepared from 6methyl - 3p - acetoxy - androst - 5 - en 17 - one (V).12 Catalytic hydrogenation of this material in
5a-ANDROST-l-ENE
249
acetic acid over palladium-on-carbon furnished the corresponding 6Prmethy 1-5a-androstane derivative. Although the initial assignment of the 6b-methyl A/B tram configuration was based on the normal steric course of hydrogenation of A5-steroids, conclusive proof was obtained by reduction of 6pmethyl-l7~-hydroxy-5a-androst-l-en-3-one (XIIa) to the known 6@-methyl-5a-androstan-17/?-01-3-one (VIIIa).lO Alkylation of the C-17 carbonyl of VIb with methyl magnesium bromide produced the l7a-methyl homolog while ethynylation followed by reduction furnished the l7a-ethyl derivative. Oxidation, bromination, and dehydrobroinination of VIb and the l7a-alkyl homologs gave the cor(?
/
CP
0
/
VIa. R=CH3C0 b.R=H
l
OH
CH3
IX
VIIa.CH3 R =CH3 b. R=C-CH C. R = CzH5
0
CH3 X
CH3 VIIIa. R = H b. R = C H j C.R=CzH5
U1,J
CH3 XIIa. R = H b. R=CH3 C. R=C,H,
-
(8) W. Bergmann, hl. Kita, and D. J. Giancala, J. Am. Chew SOC.,76, 4974 (1954). (9) H. J. Ringold, E. Batres, 0. Halpern, and E. Necoechea, J . Am. Chem. Suc., 81, 427 (1959). (IO) H. J. Ringold, E. Batres, and G. Rosenkranz, J . Org. Chem., 22, 99 (1957). (11 ) Some of the compounds in this series were reported recently by R. Mauli, H. J . Ringold, and C. Djerassi, J. Am. Chem. SOC.,82, 5494 (1960).
XIa. R = H b. K = CH,CO
(12) This compound was first prepared in these laboratories from 6-methyl-l6-dehydropregnenoloneby Dr. Walter R. Benn, unpublished work.
250
COUNSELL, KLIMSTRA, AND COLTON
responding 3-keto-A1-compounds. The 6p-methyl diol (XIa) obtained by lithium tri-t-butoxyaluminohydride reduction of Xwas converted smoothly to 6p methyl - 17p - hydroxy - 5a androst - 1en-3-one (XIIa) by manganese dioxide oxidation. Biological activity.13The assay used to determine androgenic and myotrophic activities was an adaptation14 of that employed by Eisenberg and G ~ r d a n . 'The ~ compounds were injected intramuscularly into castrate male rats and their effect on the seminal vesicles, ventral prostates and levator ani muscles compared with those produced by testosterone propionate. Table I shows the estimates of the androgenic and myotrophic potencies obtained in this assay. In contrast with earlier studies, the A l isomer of testosterone (IIa) was found to be much more active than testosterone both androgenically and myotrophically. Alkylation a t C-17 resulted in a diminution of activity which appeared to be nullified by reduction of the C-3 carbonyl group (IIId and IIIe). Methylation a t C-2 appeared to have little effect on the relative potencies; but a slight
-
-
TABLE I ANABOLIC-ANDROGENIC ACTIVITIES OF &ME Sa-ANDROST-l-ENE DEBIVATNES Compound Testosterone prbpionate Testosterone IIa IIb IIC I Id IIe IIf IIIa IIIC IlId IIIe IIIf IIIg XIIa XIIb XIIC
Androgenicu Potency
Myotrophica Potency
100
100
35 100 25 2 50 25 1 50 50. 100 100 5 2
26 200 50 5 50 50 4 40 40 50 200 2
10
10 hlhyl-l?'a-ethynyl-5a-androstan-3~,1 i'p-diol (VIIb). To a solution of diethylene glycol dimethyl ether (500 ml.) and diethylene glycol monoethyl ether (27 ml.) heated to 135' were added portion-wise under nitrogen potassium hydroxide flakes (80 g.). The mixt,ure was heated a t this temperature for 0.5 hr. with vigorous stirring and allowed to cool slowly to room temperature. The reaction vessel was surrounded with an ice-isopropyl alcohol bath and acetylene gas passed into the rapidly stirred mixture for 2.25 hr. A t this point, 6j3-methyl-5~-androstan3p-ol-l7-one (20 g.) was added as a slurry in diethylene glycol dimethyl ether (50 ml.). Stirring and acetylene addition were continued for an additional 1.5 hr. Water (250 ml.) was added to the reaction mixture, and the contents poured slowly into a mixture of ice and dilute hydrochloric acid ( l : l O , 3 1.) The yellow precipitate was removed by filtration and dissolved in ether. The ether solution was washed successively with water, dilute hydrochloric acid (1 :3), 5y0 sodium bicarbonate solution, and water. After drying the solution over a mixture of anhydrous potassium carbonate and Darco, the solvent was removed by distillation under reduced pressure. The resulting oil was crystallized from acetone to give VIIb (16.7 g.), m.p. 185-188", CY]^ -35'. Anal. Calcd for CzzHsrOz: C, 79.95; H, 10.37. Found: C, 80.07; H, 10.52. 6B-A~eethvl-l7a-elhul-6a-androstan-3~J?~-diol (VIIc). A solution oi VIIb (17.15 g.) in methanol (200 ml.) was hydrogenated in a Parr low pressure apparatus using 57, palladium-on-charcoal (1.5 9.) as catalyst. After hydrogen uptake was complete (10 min.), the catalyst was removed by filtration and washed well with methanol. The solvent wm removed from the filtrate by distillation under reduced pressure. The resulting solid residue was recrystallized from acetone-water to give VIIc (16.35 g . ) , m.p. 188.5-189.5", [CY]? - 16' (methanol). Anal. Calcd. for CaHssOt: C, 78.98; H, 11.45. Found: C, 78.75; H, 11.18. 68,I7a-DimethyE-6cu-androstan-17fi-ol~~e (VIIIb). To a solution of VIIa ( 5 g.) in acetone (100 ml.) was added standard chromium trioxide reagenP dropwise with stirring until a faint color of the reagent persisted. The excess reagent waa decomposed with isopropyl alcohol and the result(25) K. Bowden, I. hf. Heilbron, E. R. €€. Jones, and B. C. L. Weedon, J. Chcm. Soc., 39 (1946).
JANUARY
1962
DERIVATIVES OF 5a-ANDROST-l-ENE
ing solution decanted from the salts into II. mixture of ice and water. The resulting precipitate was removed by filtration and recrystallized from ethanol-water to afford VIIIb (4.0g.),m.p. 171-173", CY]^ -18.8". Anal. Calcd. for CZ1H~,O2: C, 79.19: HI 10.76. Found: C, 79.28; H, 10.66. 66- Methyl-l7a-ethyl-6a-androstan-l7~-0~-3-0ne (VIIIc). Oxidation of VIIc (14.0 g.) as described above and recrystallization of the product from ethanol-water gave VIIIc (10.5 g.), m.p. 154-156", [a]? -8". Anal. Calcd. for CaHtsOI: C 79.46; H, 10.91. Found: C, 79.07; H, 10.81. 6B-Methylda-androstan-S,17-dione (JX). Oxidation of an acetone solution of VIb (5.7 g.) with chromic acid aa described above afforded the desired &one IX (5.2 g.), m p. 169-172". Recrystallization from methanol-water gave an analytical sample, m.p. 171-173", [ a ] $81". Anal. Calcd. for CIOHmO2:C, 79.42; H, 10.00. Found: C, 79.79; H, 9.93. 6B-Methyl-6a-androat-l-en-S,l7-dion.e (X). To a solution of I X (1.07 g.) in acetic acid (25 ml.) waa added dropwise with stirring 0.608N bromine in acetic acid (12 ml.). Water waa added to the colorless solution and the resulting precipitate removed by filtration. The product, was dried in vacuo to give the crude bromo compound (1.15 g.), a portion of which (0.8 g.) waa dehydrobrominated by refluxing in collidine ( 3 ml.) for 30 min. Ether was added to the cooled solution and the mixture washed successively with 2 N hydrochloric acid, 5Q/, sodium bicarbonate solution, and water. After drying the ether solution with anhydrous pot m i u m carbonate, the solvent wm removed by distillation to give an oily residue. Adsorption of the crude product on silica gel and crystallization of the benzene-ethyl acetate ( 1 9 : l ) eluates from methanol-water gave pure XI m.p. 143-145", [a]: +log", A, 228 mp, log e 4.01. Anal. Calcd. for CmH%02:C, 79.95; H, 9.39. Found: C, 79.61; HI 9.33. 6~-Methylda-androst-l-en-3~,17p-diol (XIa). To a solution of compound X (2.5 g.) in purified tetrahydrofuran (75 ml.) waa added all a t once a solution of lithium tri-t-butoxyaluminohydride (6.0 g.) in purified tetrahydrofuran (75 ml.). The reaction was carried out under nitrogen and external cooling waa provided by an ice bath. The solution was stirred for 2 hr. and poured into ice-cold 5y0 acetic acid (500 ml.). The precipitate was collected and washed with 5% sodium bicarbonate solution and water. Recrystallizsr tion from methanol-water gave pure diol (2.3 g.), m.p. 170-173", [ala' $23". Anal. Calcd. for C&H~OI:C, 7889, H, 10.59. Found: C, 78.98; H, 10.31.
a.
253
6B-Methyl-6a-androst-I-enSB,17~-dioldiacetate (XIb). Acetylation of XIa (0.7 9.) with acetic anhydride-pyridine in the usual manner, and recrystallization of the crude product from methanol-water afforded the diacetate (0.45 g.), m.p. 111-112", [a]: +41.5". Anal. Calcd. for C I ~ H ~ ~ O C,, :74.19; H, 9.34. Found: C, 74.03; H, 9.10. 66-Methyl-1'?,Y-hyd~oxy-6a-androat-l-end-cme (XIIs). A mixture of XIa (0.5 g.), isopropyl alcohoP(20 ml.), and manganese dioxide27 (4.5 g.) was stirred at room temperature for 18 hr. The mixture was filtered and water added to the filtrate until the solution became slightly turbid. Refrigeration and collection of the crystalline product afforded A, pure XIIa (0.35 g.), m.p. - 182-182.5",. [a]:: . _ _ +19", = 527.5 mp, log e 3.99.' Anal. Calcd. for CmHmOp: C, 79.42; H, 10.00. Found: C. 79.27: H, 9.99. 'Catalytic' hydrogenation of XIIa. A solution of XIIa (82 mg.) in ethanol (15 ml.) was hydrogenated at 29.5" and ahmospheric pressure over 5% palladium-on-carbon (30 mg.). After hydrogen uptake ceased (3 hr.), the catalyst was removed by filt,ration and washed with ethanol. Removal of the solvent from the filtrate and recrystallization of the resulting solid from acetone-hexane gave 68-methyl-5aandrostan-17p-ol-3-one (VIIIa), m.p. 201-203.5" (reportedlo m.p. 203-205"), infrared"": ' :X: 2.75,3.37, 5.83 p. 6,5',1 7a-Dimethyl-l7~-hydroxydu-androst-l -en-3-one (XIIb) was prepared by bromination of VIIIb in dimethylformamide and the crude product dehydrobrominated in the usual manner. Purification of the product by chromatography on silica gel gave pure XIIb, m.p. 175-177", [a]? +1.5", ,A, 228.5 mp, log e 4.00. Anal. Calcd. for CzlHJIO1:C, 79.69; H, 10.19. Found: C, 79.71; H, H, 10.23. 6p-Methyl-1 ra-ethyl-178-hydroxyda-androst-1-en-3-on e (XIIc) was prepared by bromination of VIIIc in dimethylformamide and the crude product dehydrobrominated in the usual manner. Purification of the product by the bisulfite method gave pure XIIc, m.p. 165-168' [a]; +7" Amax 228.5 mp, log E 3.96. Anal. Calcd. for CIPH~OI: C, 79.95; H, 10.37. Found: C, 79.78; H, 10.56. CHICAGO 80, ILL. (26) We are grateful to Dr. C. G. Rergstrom for pointing out the utility of isopropyl alcohol in this reaction. ' (27) 0. Mancera, G. Rosenkranz, and F. Sondheimer, J. Chem. SOC.,2189 (1953).
